Updated: Tue Nov 4 17:59:00 2014

Introduction

Neurospheres Cortex vs GE

  • Genetic mutations to epigenetic regulators and epigenomic alterations are amongst the earliest events in brain cell transformation. PMID: 18772396
  • Defining the epigenomic landscape of normal human brain cells is an important first step towards defining the degree of epigenomic deregulation associated with transformation.
  • Neurospheres provide a powerful model to study neural stem cells, which are thought to be population from which malignant clones arise.
  • Little is known about epigenetic differences that define neurospheres emerged from distinct brain regions.

MZ twins

  • The genomes of monozygotic twins are genetically identical but epigenetically distinct providing evidence for the influence of environment on the phenotype.
  • When these epigenetic differences arise during development and their consequence is still unknown.

Results

Neurospheres Cortex derived vs GE derived

Asymmetry between Cortex and GE UMRs

  • On average, there are , 4.31-fold enrichment in total UMR frequency in Cortex compared to GE, 4.07 in HuFNSC02, and 5.31 in HuFNSC04.
  • The asymmetry appears to be global, in all chromosomes. It is reproduced in the two individuals, and supported in MeDIP UMRs.
  • Single CpG level differential methylation is symmetric, but the asymmetry on UMR level can be reproduced with different cutoffs. However, there are no apparent differences in UMR length between Cortex and GE, suggesting that there are more orphan GE UM CpGs that was not able to form UMRs than in Cortex.
Sample Total.DMR Hyper.DMR Hypo.DMR
Cortex-HuFNSC02_GE-HuFNSC02 2178 420 1758
Cortex-HuFNSC04_GE-HuFNSC04 646 91 555

GREAT analysis on Cortex and GE UMRs show brain development terms

  • UMRs in both Cortex and GE show enrichment in neuron fate commitment biological process, transcriptional regulation activities, Homeobox protein domain, and abnormal brain development mouse phenotype.
  • Cortex UMRs are also enriched in forebrain regionalization and pattern specification processes.
  • GREAT enriched terms are also supported by MeDIP.
  • In HuFNSC04, Cortex UMRs show abnormal brain development in Mouse Phenotype, but are also enriched in kidney-related processes.

UMR breakdown in chromatin states - TBC

  • Pending: ChromHMM

DE genes with proximal UMRs show key factors in brain development

  • On average, there are 40 UMRs proximally associated with protein-coding genes (TSS +/- 1500bp), 2.85% of all UMRs.
  • There are average 8 proximal UMRs associated with DE genes, 19.88% of all proximal UMRs, much lower than observed in breast and supported by MeDIP, maybe more UMRs in enhancers? (ChromHMM). Among them, there are 46.67% unique DE genes change in the same direction as the UMRs, also lower than observed in breast and supported by MeDIP.
  • The intersect between two individuals are significant in Cortex UMRs. There are 3 DE genes with proximal Cortex UMR that are shared by both individual:
    • GFAP: Glial Fibrillary Acidic Protein. It is used as a marker to distinguish astrocytes from other glial cells during development. Reported associated with many brain disease, including alexanders disease, PMID: 11567214; gliomas, UP reduce tumor and induce differentiation, PMID: 15498217, and astrocytoma, tumor suppressor PMID: 8339269 etc.
    • NFIX: Nuclear Factor I/X (CCAAT-Binding Transcription Factor). Also supported by MeDIP. A transcription factor that binds the palindromic sequence in viral and cellular promoters, capable of activating transcription and replication. It is essential for the development of a number of organ systems including brain and bone, e.g. severe neuroanatomical defects (may function in the repression of neural stem cell proliferation or in cell migration) PMID: 18477394, PMID: 19058033.
    • FEZF1: FEZ Family Zinc Finger 1 (ZNF312B). Transcription repressor. Involved in the axonal projection and proper termination of olfactory sensory neurons. Regulates non-cell-autonomously the layer formation of the olfactory bulb development and the interneurons. May be required for correct rostral migration of the interneuron progenitors. DNA demethylation and histone acetylation in its promoter activates its expression and oncogene effect in gastric cancer PMID: 19318583. We observe DN-regulation in Cortex and Cortex UMR though.
  • There are no intersect in pc genes with proximal GE UMRs between the two individual.
  • There are no significant DAVID enrichment terms due to the small number of genes.
proximal.UMRs unique.genes DE.DMRs unique.DE.genes same.direction
GE_UMRs-HuFNSC02 17 17 4 4 1
Cortex_UMRs-HuFNSC02 53 52 15 14 8
GE_UMRs-HuFNSC04 9 11 3 3 2
Cortex_UMRs-HuFNSC04 82 84 10 9 3

DE genes with proximal Cortex UMRs
  • HuFNSC02
name description DE
CD58 CD58_molecule UP
RGS10 regulator_of_G-protein_signaling_10 UP
ADM adrenomedullin UP
NKX2-1 NK2_homeobox_1 DN
OTX2 orthodenticle_homeobox_2 DN
USP43 ubiquitin_specific_peptidase_43 DN
GFAP glial_fibrillary_acidic_protein UP
NFIX nuclear_factor_I/X_(CCAAT-binding_transcription_factor) UP
GAD1 glutamate_decarboxylase_1_(brain,_67kDa) DN
FZD7 frizzled_family_receptor_7 UP
FZD5 frizzled_family_receptor_5 DN
CXCR7 chemokine_(C-X-C_motif)receptor7 DN
ZAR1 zygote_arrest_1 UP
FEZF1 FEZ_family_zinc_finger_1 DN
  • HuFNSC04
name description DE
FAM5C family_with_sequence_similarity_5,memberC UP
STXBP6 syntaxin_binding_protein_6_(amisyn) DN
GFAP glial_fibrillary_acidic_protein UP
NFIX nuclear_factor_I/X_(CCAAT-binding_transcription_factor) UP
MEIS1 Meis_homeobox_1 DN
FSIP2 fibrous_sheath_interacting_protein_2 DN
INSM1 insulinoma-associated_1 DN
FEZF1 FEZ_family_zinc_finger_1 DN
C9orf172 chromosome_9_open_reading_frame_172 DN
DE genes with proximal GE UMRs
  • HuFNSC02
name description DE
PAX6 paired_box_6 UP
TMEM132B transmembrane_protein_132B UP
PID1 phosphotyrosine_interaction_domain_containing_1 DN
ZIC3 Zic_family_member_3 UP
  • HuFNSC04
name description DE
MN1 meningioma_(disrupted_in_balanced_translocation)_1 DN
PNCK pregnancy_up-regulated_non-ubiquitously_expressed_CaM_kinase UP

UMR distal associated genes - TBC

  • PENDING

Asymmetry in overlapping UMRs with TFBSs

  • Overlap UMRs with transcription factor binding sites and count No. of overlapping TFBSs for each TF showed similar asymmetry between Cortex and GE in both individuals, with TFBSs enriched in Cortex UMRs for most TFs. Opposite trend in HuFNSC01 MeDIP.
  • However, in general, the correlation of TFBS Cortex UMR vs GE UMR fold change between the two individual is quite low, only 0.2. Same in MeDIP.
  • There are 15 TFs that are at least 3-fold enriched in TFBSs overlapping Cortex UMRs compared to GE UMRs as shown below.
TF Cortex02UMR GE02UMR Ratio02 Cortex04UMR GE04UMR Ratio04
TAF7 12 4 3.00 10 1 10.00
TFAP2A 30 10 3.00 20 3 6.67
USF1 67 21 3.19 31 6 5.17
ELF1 45 14 3.21 39 5 7.80
GATA2 116 36 3.22 43 4 10.75
ESR1 55 17 3.24 9 3 3.00
CTCF 197 56 3.52 105 16 6.56
TFAP2C 32 9 3.56 23 5 4.60
MYC 133 36 3.69 84 5 16.80
FOXA1 89 24 3.71 26 5 5.20
TCF7L2 82 22 3.73 47 4 11.75
ZNF263 31 8 3.88 23 5 4.60
E2F1 35 9 3.89 38 5 7.60
TAL1 16 4 4.00 5 1 5.00
GATA3 63 8 7.88 18 1 18.00

DE genes between Cortex and GE are enriched in neuron development and cell migration

  • On average, there are 860 genes differentially expressed between cortex and GE, among them, 454 are upregulated in cortex, and 406 are downregulated.
  • 382 Cortex up-regulated genes, and 456 GE up-regulated genes are shared by at least two individuals.
  • DAVID enrichment analysis show significant enrichment in neuronal development and cell migration terms, GE up-regulated genes are enriched in EGF-related protein domains as well.
  • There are 22 Cortex up-regulated genes, and 26 GE up-regulated genes shared by all four individuals.
  • Brain / neuron development related Cortex up-regulated genes (shared by all):
    • SLC1A6: Solute Carrier Family 1 Member 6. Glutamate transporter in cerebellar Purkinje neurons, related to traumatic brain injury.
    • PCDH20: Protocadherin 20. Although its specific function is undetermined, the cadherin-related neuronal receptor is thought to play a role in the establishment and function of specific cell-cell connections in the brain.
    • GFAP: see above.
    • ZIC5: Zic Family Member 5. Essential for neural crest development, converting cells from an epidermal fate to a neural crest cell fate, associated with meningiomas PMID: 20199689.
    • ZIC2: Zic Family Member 2. Related to structural anomaly of the forebrain, and neural tube defects.
    • NEFM: Neurofilament, Medium Polypeptide. Neurofilaments comprise the axoskeleton and functionally maintain neuronal caliber. They may also play a role in intracellular transport to axons and dendrites. This protein is commonly used as a biomarker of neuronal damage.
    • UNC5C: Unc-5 Homolog C. Receptor for netrin required for axon guidance. Mediates axon repulsion of neuronal growth cones in the developing nervous system upon ligand binding.
    • ADAM19: ADAM Metallopeptidase Domain 19. Involved in neurogenesis, cell migration, cell adhesion, cell-cell and cell-matrix interactions, serves as a marker for dendritic cell differentiation. UP in primary brain tumors PMID: 16772875.
  • Brain / neuron development related GE up-regulated genes (shared by all):
    • VAX1: Ventral Anterior Homeobox 1. Transcription factor that may function in dorsoventral specification of the forebrain. Required for axon guidance and major tract formation in the developing forebrain.
    • NRXN3: Neurexin 3. Neuronal cell surface protein that may be involved in cell recognition and cell adhesion.
    • FEZF1: see above.
    • SIX3: SIX Homeobox 3. Plays a role in eye development.
    • ODZ1: Odz, Odd Oz/Ten-M Homolog 1. Involved in neural development, regulating the establishment of proper connectivity within the nervous system.
    • EPHA3: EPH Receptor A3. Involved in short-range contact-mediated axon guidance. UP in glioblastoma PMID: 23410976.
    • OTX2: Orthodenticle Homeobox 2. Plays a role in the development of the brain and the sense organs, also influences the proliferation and differentiation of dopaminergic neuronal progenitor cells during mitosis. UP in medulloblastomas PMID: 20028867.
    • ASTN1: Astrotactin 1. A neuronal adhesion molecule required for glial-guided migration of young postmitotic neuroblasts in cortical regions of developing brain, including cerebrum, hippocampus, cerebellum, and olfactory bulb (Fink et al., 1995).
UP DN DE
HuFNSC01 403 508 911
HuFNSC02 588 640 1228
HuFNSC03 447 227 674
HuFNSC04 378 249 627
DE name description
UP SLC1A6 solute_carrier_family_1_(high_affinity_aspartate/glutamate_transporter),member6
UP PCDH20 protocadherin_20
UP C9orf64 chromosome_9_open_reading_frame_64
UP GFAP glial_fibrillary_acidic_protein
UP ZIC5 Zic_family_member_5
UP NEFM neurofilament,mediumpolypeptide
UP QPCT glutaminyl-peptide_cyclotransferase
UP UNC5C unc-5_homolog_C_(C._elegans)
UP ZIC2 Zic_family_member_2
UP XKR8 XK,Kellblood_group_complex_subunit-related_family,member8
UP PYGL phosphorylase,glycogen,liver
UP SERINC2 serine_incorporator_2
UP KIAA1239 KIAA1239
UP B3GALT1 UDP-Gal:betaGlcNAc_beta_1,3-galactosyltransferase,polypeptide1
UP CCDC48 coiled-coil_domain_containing_48
UP NOS1AP nitric_oxide_synthase_1_(neuronal)adaptorprotein
UP ADAM19 ADAM_metallopeptidase_domain_19
UP H1F0 H1_histone_family,member0
UP ZIC3 Zic_family_member_3
UP NT5E 5’-nucleotidase,ecto(CD73)
UP SYNM synemin,intermediatefilament_protein
UP C1orf226 chromosome_1_open_reading_frame_226
DE name description
DN DPPA4 developmental_pluripotency_associated_4
DN VAX1 ventral_anterior_homeobox_1
DN SHISA6 shisa_homolog_6_(Xenopus_laevis)
DN NRXN3 neurexin_3
DN FGFR2 fibroblast_growth_factor_receptor_2
DN ST8SIA2 ST8_alpha-N-acetyl-neuraminide_alpha-2,8-sialyltransferase_2
DN SLC32A1 solute_carrier_family_32_(GABA_vesicular_transporter),member1
DN FEZF1 FEZ_family_zinc_finger_1
DN AJAP1 adherens_junctions_associated_protein_1
DN ZNF703 zinc_finger_protein_703
DN VSIG10L V-set_and_immunoglobulin_domain_containing_10_like
DN SIX3 SIX_homeobox_3
DN SORL1 sortilin-related_receptor,L(DLRclass)Arepeats_containing
DN LMO1 LIM_domain_only_1_(rhombotin_1)
DN ODZ1 odz,oddOz/ten-m_homolog_1_(Drosophila)
DN EPHA3 EPH_receptor_A3
DN TIMP3 TIMP_metallopeptidase_inhibitor_3
DN CAMK2N1 calcium/calmodulin-dependent_protein_kinase_II_inhibitor_1
DN EEF1A2 eukaryotic_translation_elongation_factor_1_alpha_2
DN OTX2 orthodenticle_homeobox_2
DN CHL1 cell_adhesion_molecule_with_homology_to_L1CAM_(close_homolog_of_L1)
DN PCSK1N proprotein_convertase_subtilisin/kexin_type_1_inhibitor
DN LMO2 LIM_domain_only_2_(rhombotin-like_1)
DN ASTN1 astrotactin_1
DN BASP1 brain_abundant,membraneattached_signal_protein_1
DN ADCYAP1R1 adenylate_cyclase_activating_polypeptide_1_(pituitary)receptortype_I

Isoforms between Cortex and GE are enriched in cell signaling proteins

  • On average, 2054 genes are identified as isoforms between cortex and GE in each individual. 2352 genes are shared in at least two individuals.
  • Individual specific isoforms between cortex and GE have no significantly enriched terms, suggesting they are more likely random events without biological functions.
  • Isoforms shared by at least two individuals are enriched in terms related to cellular signaling. InterPro shows enrichment in EGF protein domain.
DE_genes DE_exons with_expressed_genes isoform_exons exclude_DE_genes isoform_genes
cortex01_GE01_summary 911 32372 18968 8440 7962 2447
cortex02_GE02_summary 1228 35196 21880 8163 7374 2298
cortex03_GE03_summary 674 29617 13746 6401 6022 2086
cortex04_GE04_summary 627 22386 11253 4323 4259 1386

Intron retention between Cortex and GE - TBC

  • PENDING

mCpG is a stable mark for exon transcription during development

  • mCpG at cassette exon boundaries has similar pattern to expressed in both exons.
  • mCpG for cassette exons between cortex and GE neurospheres shows no significant differences.
  • Results from both WGBS (HuFNSC02 & HuFNSC04) and MeDIP (HuFNSC01 & HuFNSC02) support the assumption that mCpG is a stable mark for exon transcription during development.
  • mCpG exon marking is established between neurospheres. Needs further validation against H1.

H3K36me3 in exon bodies - normalization issue?

  • H3K36me3 in expressed in both / not expressed exons shows no significant differences between HuFNSC01 and HuFNSC02.
  • H3K36me3 in cassette exons in HuFNSC01 are enriched in GE compared to cortex. However, it is not reproduced in HuFNSC02, where there is no significant differences between cortex and GE. Not sure what to make of this. Are there any potential bias?

MZ twins

UMR asymmetry between MZ twins in Brain and Cortex

  • There is an asymmetry between UMRs in HuFNSC01 and HuFNSC02 in the Brain and Cortex neurosphere, but not in GE neurosphere. Fold change in UMR frequency HuFNSC02/HuFNSC01 in Brain is 2.41, in Cortex is 1.83, and in GE is 0.899.
Sample Total.DMR Hyper.DMR Hypo.DMR
Brain-HuFNSC01_Brain-HuFNSC02 4472 2750 1722
Cortex-HuFNSC01_Cortex-HuFNSC02 3161 1758 1403
GE-HuFNSC01_GE-HuFNSC02 2716 1136 1580

GREAT analysis on MZ UMRs are enriched in Homeobox and brain development

  • Homeobox protein domain is enriched in all UMR lists.
  • Brain development related biological processes are enriched in most lists.

UMR breakdown in chromatin states - TBC

  • Pending: ChromHMM

MZ UMRs in Brain have less CpGs in CGIs

  • Majority of UMR CpGs overlap with genebody, on average 62.59%. And UMR CpGs are 3-fold enriched in promoter regions, with on average 34.29% UMR CpGs are in promoter regions, 39.54% of CpGs in UMRs overlap with CGIs, 5.34-fold than expected by random.
  • Brain seems to have less CGI/promoter UMRs.

DE genes with proximal UMRs between MZ are cell type specific

  • On average, there are 405 UMRs proximally (TSS +/- 1500bp) associated with protein-coding genes, 11.75% of all UMRs, similar to UMRs between Cortex and GE.
  • On average, there are 17 proximal UMRs associated with DE genes, 4.24% of all proximal DMRs, much less than UMRs between Cortex and GE (less functional?). Among them, there are 52.53% unique DE genes change in the same direction as the UMRs, similar to UMRs between Cortex and GE.
  • Proximal UMR genes are mostly cell type specific, but the overlap is still statistically significant.
  • No overlap between Brain and Cortex for proximal HuFNSC01 UMR DE genes, only one for HuFNSC02, neuropeptide_Y.
  • HuFNSC01 UMR DE genes associated with brain development:
    • WNT pathway: SFRP2, WNT3.
    • EMX2: Empty Spiracles Homeobox 2. Transcription factor. Acts to generate the boundary between the roof and archipallium in the developing brain. May function in combinations with OTX1/2 to specify cell fates in the developing central nervous system.
    • MDGA1: MAM Domain-Containing Glycosylphosphatidylinositol Anchor Protein 1. Required for radial migration of cortical neurons in the superficial layer of the neocortex.
    • VAX1: see above.
    • DLX1: Distal-Less Homeobox 1. Play a role in the control of craniofacial patterning and the differentiation and survival of inhibitory neurons in the forebrain.
    • OLIG1: Oligodendrocyte Transcription Factor 1. Promotes formation and maturation of oligodendrocytes, especially within the brain. Associated with human glial brain tumors PMID: 11526205.
  • HuFNSC02 UMR DE genes associated with brain development:
    • WNT pathway: SFRP1, SFRP2, WNT7A.
    • EPHA8: EPH Receptor A8. Plays a role in short-range contact-mediated axonal guidance during development of the mammalian nervous system.
    • NRGN: Neurogranin (Protein Kinase C Substrate, RC3). Acts as a “third messenger” substrate of protein kinase C-mediated molecular cascades during synaptic development and remodeling.
    • C1QL4: Complement Component 1, Q Subcomponent-Like 4. May regulate the number of excitatory synapses that are formed on hippocampus neurons.
    • SNCB: Synuclein, Beta. May play a role in neuronal plasticity.
    • SEMA3D: Sema Domain, Immunoglobulin Domain (Ig), Short Basic Domain, Secreted, (Semaphorin) 3D. Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations.
    • VGF: May be involved in the regulation of cell-cell interactions or in synatogenesis during the maturation of the nervous system.
DMRs unique.genes DE.DMRs unique.DE.genes same.direction
Brain01_Brain02_hyper 432 473 26 26 10
Brain01_Brain02_hypo 367 409 20 20 13
Cortex01_Cortex02_hyper 587 659 30 28 17
Cortex01_Cortex02_hypo 316 342 24 22 11
GE01_GE02_hyper 369 402 1 1 1
GE01_GE02_hypo 361 398 2 2 0

HuFNSC01 UMRs proximal associated DE genes
CellType name description DM DE
Brain SAMD11 sterile_alpha_motif_domain_containing_11 hypo UP
Brain ITGA8 integrin,alpha8 hypo UP
Brain EMX2 empty_spiracles_homeobox_2 hypo DN
Brain ARNTL2 aryl_hydrocarbon_receptor_nuclear_translocator-like_2 hypo UP
Brain CPNE8 copine_VIII hypo DN
Brain PCDH17 protocadherin_17 hypo DN
Brain Putative_3-phosphoinositide-dependent_protein_kinase_2 hypo UP
Brain CPNE7 copine_VII hypo UP
Brain PKDREJ polycystic_kidney_disease_(polycystin)andREJ_homolog_(sperm_receptor_for_egg_jelly_homolog,seaurchin) hypo UP
Brain THRB thyroid_hormone_receptor,_beta hypo UP
Brain SFRP2 secreted_frizzled-related_protein_2 hypo DN
Brain IER3 immediate_early_response_3 hypo UP
Brain ZBTB12 zinc_finger_and_BTB_domain_containing_12 hypo UP
Brain MDGA1 MAM_domain_containing_glycosylphosphatidylinositol_anchor_1 hypo DN
Brain EPHA1 EPH_receptor_A1 hypo UP
Brain Protein_kinase-like_protein_SgK196 hypo UP
Brain TOX thymocyte_selection-associated_high_mobility_group_box hypo DN
Brain NCOA2 nuclear_receptor_coactivator_2 hypo UP
Brain GPR64 G_protein-coupled_receptor_64 hypo UP
Brain TRPC5 transient_receptor_potential_cation_channel,subfamilyC,member5 hypo DN
Cortex ELTD1 EGF,latrophilinand_seven_transmembrane_domain_containing_1 hypo UP
Cortex CRABP2 cellular_retinoic_acid_binding_protein_2 hypo DN
Cortex SPOCK2 sparc/osteonectin,cwcvand_kazal-like_domains_proteoglycan_(testican)_2 hypo DN
Cortex VAX1 ventral_anterior_homeobox_1 hypo UP
Cortex LRRC10B leucine_rich_repeat_containing_10B hypo DN
Cortex NDUFA4L2 NADH_dehydrogenase_(ubiquinone)1alpha_subcomplex,4-like2 hypo DN
Cortex FOXO1 forkhead_box_O1 hypo UP
Cortex C16orf74 chromosome_16_open_reading_frame_74 hypo DN
Cortex WNT3 wingless-type_MMTV_integration_site_family,member3 hypo DN
Cortex CHST9 carbohydrate_(N-acetylgalactosamine_4-0)sulfotransferase9 hypo UP
Cortex CD97 CD97_molecule hypo DN
Cortex RNF144A ring_finger_protein_144A hypo UP
Cortex DLX1 distal-less_homeobox_1 hypo UP
Cortex OLIG1 oligodendrocyte_transcription_factor_1 hypo UP
Cortex SCUBE1 signal_peptide,CUBdomain,EGF-like1 hypo DN
Cortex GRIA1 glutamate_receptor,ionotropic,AMPA_1 hypo DN
Cortex MMD2 monocyte_to_macrophage_differentiation-associated_2 hypo UP
Cortex SEMA3E sema_domain,immunoglobulindomain_(Ig),shortbasic_domain,secreted,(semaphorin)_3E hypo DN
Cortex VIPR2 vasoactive_intestinal_peptide_receptor_2 hypo DN
Cortex AP1S2 adaptor-related_protein_complex_1,sigma2_subunit hypo DN
Cortex SH3KBP1 SH3-domain_kinase_binding_protein_1 hypo UP
GE FAM5B family_with_sequence_similarity_5,memberB hypo DN
GE DLL1 delta-like_1_(Drosophila) hypo DN
HuFNSC02 UMRs proximal associated DE genes
CellType name description DM DE
Brain EPHA8 EPH_receptor_A8 hyper UP
Brain IFI6 interferon,alpha-inducibleprotein_6 hyper DN
Brain BMP8A bone_morphogenetic_protein_8a hyper UP
Brain ALX3 ALX_homeobox_3 hyper UP
Brain NRGN neurogranin_(protein_kinase_C_substrate,_RC3) hyper UP
Brain C1QL4 complement_component_1,qsubcomponent-like_4 hyper UP
Brain CSRP2 cysteine_and_glycine-rich_protein_2 hyper DN
Brain TTYH2 tweety_homolog_2_(Drosophila) hyper DN
Brain DTNB dystrobrevin,_beta hyper UP
Brain CXCR7 chemokine_(C-X-C_motif)receptor7 hyper UP
Brain VHL von_Hippel-Lindau_tumor_suppressor hyper UP
Brain EPHA6 EPH_receptor_A6 hyper UP
Brain CAMK2N2 calcium/calmodulin-dependent_protein_kinase_II_inhibitor_2 hyper UP
Brain TBC1D1 TBC1_(tre-2/USP6,BUB2,cdc16)domainfamily,member1 hyper DN
Brain NMU neuromedin_U hyper DN
Brain FSTL5 follistatin-like_5 hyper DN
Brain CARTPT CART_prepropeptide hyper DN
Brain EDIL3 EGF-like_repeats_and_discoidin_I-like_domains_3 hyper UP
Brain CCDC90A coiled-coil_domain_containing_90A hyper UP
Brain LOC401296_proteinUncharacterized_protein hyper UP
Brain NPY neuropeptide_Y hyper UP
Brain SFRP1 secreted_frizzled-related_protein_1 hyper DN
Brain TOX thymocyte_selection-associated_high_mobility_group_box hyper DN
Brain ENTPD2 ectonucleoside_triphosphate_diphosphohydrolase_2 hyper UP
Brain KLHL4 kelch-like_4_(Drosophila) hyper UP
Brain SOX3 SRY_(sex_determining_region_Y)-box_3 hyper DN
Cortex DMRTA2 DMRT-like_family_A2 hyper DN
Cortex NPR1 natriuretic_peptide_receptor_A/guanylate_cyclase_A_(atrionatriuretic_peptide_receptor_A) hyper UP
Cortex PTPRE protein_tyrosine_phosphatase,receptortype,_E hyper DN
Cortex PDE3B phosphodiesterase_3B,_cGMP-inhibited hyper UP
Cortex LDHA lactate_dehydrogenase_A hyper DN
Cortex GNG3 guanine_nucleotide_binding_protein_(G_protein),gamma3 hyper DN
Cortex METTL7B methyltransferase_like_7B hyper DN
Cortex NME3 non-metastatic_cells_3,proteinexpressed_in hyper DN
Cortex GRIN2A glutamate_receptor,ionotropic,N-methyl_D-aspartate_2A hyper UP
Cortex NOL3 nucleolar_protein_3_(apoptosis_repressor_with_CARD_domain) hyper DN
Cortex NR1D1 nuclear_receptor_subfamily_1,groupD,member1 hyper DN
Cortex IGFBP4 insulin-like_growth_factor_binding_protein_4 hyper UP
Cortex TBX2 T-box_2 hyper UP
Cortex LBH limb_bud_and_heart_development_homolog_(mouse) hyper DN
Cortex INSIG2 insulin_induced_gene_2 hyper DN
Cortex WNT7A wingless-type_MMTV_integration_site_family,member7A hyper DN
Cortex SNCA synuclein,alpha(non_A4_component_of_amyloid_precursor) hyper UP
Cortex NPNT nephronectin hyper DN
Cortex SFRP2 secreted_frizzled-related_protein_2 hyper UP
Cortex PRR16 proline_rich_16 hyper UP
Cortex STC2 stanniocalcin_2 hyper DN
Cortex SNCB synuclein,_beta hyper DN
Cortex ITPR3 inositol_1,4,5-trisphosphate_receptor,type3 hyper UP
Cortex NPY neuropeptide_Y hyper DN
Cortex SEMA3D sema_domain,immunoglobulindomain_(Ig),shortbasic_domain,secreted,(semaphorin)_3D hyper UP
Cortex VGF VGF_nerve_growth_factor_inducible hyper UP
Cortex CAV1 caveolin_1,caveolaeprotein,_22kDa hyper UP
Cortex SHROOM2 shroom_family_member_2 hyper UP
GE DBC1 deleted_in_bladder_cancer_1 hyper DN

UMR distal associated genes - TBC

  • PENDING

Overlapping UMRs with TFBSs show asymmetric in Brain and Cortex

  • Overlap UMRs with transcription factor binding sites and count No. of overlapping TFBSs for each TF showed similar asymmetry in Brain and Cortex, but is symmetric in GE. The correlation between Brain and Cortex is also very low, 0.15.
  • With 2-fold change cutoff, there are 18 TFs enriched in HuFNSC02 in both Brain and Cortex.
TF Brain.hypo Brain.hyper Ratio.Brain Cortex.hypo Cortex.hyper Ratio.Cortex
ESRRA 1 7 0.14 1 5 0.20
TAL1 10 42 0.24 9 34 0.26
HNF4A 15 49 0.31 11 27 0.41
HNF4G 13 41 0.32 10 29 0.34
ZNF217 6 17 0.35 6 14 0.43
EBF1 49 130 0.38 48 98 0.49
FOS 47 123 0.38 43 118 0.36
GATA1 31 73 0.42 32 80 0.40
HSF1 3 7 0.43 2 6 0.33
FOXA2 26 60 0.43 14 36 0.39
GATA3 35 80 0.44 22 52 0.42
GATA2 58 130 0.45 39 96 0.41
STAT3 41 91 0.45 26 87 0.30
CEBPD 22 48 0.46 23 64 0.36
SMC3 71 150 0.47 62 144 0.43
NR3C1 46 95 0.48 39 90 0.43
RXRA 23 47 0.49 16 35 0.46
HDAC6 1 2 0.50 2 11 0.18

DE genes are cell type specific

  • On average, there are 470 DE genes across three cells types.
  • Majority of DE genes are cell type specific, only 98 are shared between any two cell types.
  • DE genes in Brain is asymmetric, maybe due to cell heterogenity?
  • There are much fewer DE genes in GE.
  • DAVID enrichment analysis between MZ twins in brain and cortex show similar GO term in brain development, but there is no significantly enriched terms in GE.
UP DN DE
brain01_brain02 461 181 642
cortex01_cortex02 248 348 596
GE01_GE02 99 74 173

name description Brain Cortex GE
LMO1 LIM_domain_only_1_(rhombotin_1) UP DN DN
CXCR7 chemokine_(C-X-C_motif)receptor7 UP UP DN
SPP1 secreted_phosphoprotein_1 UP DN DN
NPY neuropeptide_Y UP DN DN
COL2A1 collagen,typeII,alpha1 DN UP UP
BCL6 B-cell_CLL/lymphoma_6 DN DN DN

Isoforms between MZ are enriched in cell signaling in neurospheres and immune response in Brain

  • On average, 2617 genes are identified as isoforms between HuFNSC01 and HuFNSC02 in each cell type. 796 genes are shared by all three cell types.
  • On average, 1724 genes are identified as isoforms between HuFNSC03 and HuFNSC04 in each cell type. 927 genes are shared between two cell types.
  • Different regions on the Venn diagram have no significantly enriched terms.
  • Isoforms between HuFNSC01 and HuFNSC02 in neurospheres show similar terms, related to cell signaling, and blood cell development in brain.
DE_genes DE_exons with_expressed_genes isoform_exons exclude_DE_genes isoform_genes
brain01_brain02_summary 642 32138 16302 8980 8542 2902
cortex01_cortex02_summary 596 26983 15554 7618 7445 2454
GE01_GE02_summary 173 23810 12862 7402 7351 2495
cortex03_cortex04_summary 642 26826 12185 5818 5479 1994
GE03_GE04_summary 545 24752 12223 4582 4422 1454

Intron retention in MZ twins - TBC

  • PENDING

Methods

DMR identification

WGBS

  • Identify DM CpGs
    • methyl_diff one-sided p-value \(\le\) 0.005
    • delta fractional methylation \(\ge\) 0.5
    • fractional methylation of one sample \(\ge\) 0.75
  • Collapse DM CpGs into DMRs
    • adjacent DM CpGs have the same DM status;
    • distance between adjacent CpGs (size) \(\le\) 300bp;
    • No. of CpGs within each DMR \(\ge\) 3.

MeDIP

  • DM CpG identification:
    • delta fractional methylation \(\ge\) 0.6
    • fractional methylation of one sample \(\ge\) 0.75
  • Collapse DM CpGs into DMRs:
    • adjacent CpGs have the same DM status;
    • distance between adjacent CpGs \(\le\) 300bp;
    • No. of CpGs within each DMR \(\ge\) 4.

Differential gene expression with DEfine

  • FDR = 0.01
  • Minimum sum of RPKM (rmin) = 0.005
  • Minimum sum of coverage (Nmin) = 25

Isoform identification and junction validation

  • DEfine on exons: FDR = 0.01
  • Exon expressed in one sample (\(\ge\) 10% gene RPKM) and not expressed in the other (\(\le\) 1% gene RPKM)
  • Gene is not DE: DEfine FDR = 0.01
  • Gene is expressed in both samples: gene RPKM > 0.01
  • Validation: For each isoform exon in the previous pairwise comparison
    • Find junctions associated with this exon with enough coverage, i.e. sum of junction coverage of two samples \(\ge\) 1
    • Identify junctions that RPKM change in the same direction as the exon
    • Junction RPKM > 0.1 in one sample and < 0.1 in the other

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